WO2015184983A1 - Rapamycin derivative, preparation method therefor, and pharmaceutical composition and uses thereof - Google Patents

Abstract

The present invention relates to the field of pharmaceutical chemicals. Provided are a rapamycin derivative represented by formula I, a preparation method therefor, and a pharmaceutical composition and uses thereof. The compounds of the present invention alleviate defects of rapamycin in water solubility and metabolic properties, and a part of the compounds are superior to rapamycin in the aspect of antitumor activity in vitro, have less toxicity to normal cells than rapamycin and have good druggability.

Description

Rapamycin derivative, preparation method thereof, pharmaceutical composition thereof and use thereof Technical field

The invention belongs to the field of medicine and chemical industry, and relates to a rapamycin derivative, a preparation method thereof, a pharmaceutical composition thereof and use thereof.

Background technique

Rapamycin, also known as sirolimus, was isolated from 1975 by Veniza et al. from Streptomyces hygroscopicus. In 1989, rapamycin entered the clinic as a novel immunosuppressant and was launched in 1999. Later, the inhibitory effect of rapamycin on the proliferation of T lymphocytes led to its use in anti-tumor cell therapy, and found that it also has good anti-tumor activity. This drug has been developed as an anticancer drug by Wyeth. Coming soon into the clinic.

The site of action of rapamycin in humans is mTOR (mammalian target of rapamycin); mTOR is an important component of the PI3K-Akt-mTOR signaling pathway. The PI3K-Akt-mTOR signaling pathway promotes the proliferation of tumor cells and enhances the ability of tumor cells to infiltrate and metastasize through regulation of tumor cell proliferation and apoptosis-related signaling pathways. On the other hand, through the tumor cells The influence of apoptosis-related proteins promotes activation of endogenous apoptosis inhibitors and/or inhibits the expression and activation of apoptosis-associated protein kinases and inhibits apoptosis of tumor cells. Therefore, PI3K-Akt-mTOR signaling pathway may be the core regulatory link of tumorigenesis and development, which makes mTOR a key target for gene therapy of tumors.

mTOR is mainly present in the form of two complexes in animals, namely mTORC1 and mTORC2. mTORC1 (mTOR complex 1) consists of mTOR, raptor (regulatory associated protein of mTOR), mLST8, PRAS40 (Proline-rich AKT Substrate 40kDa); mTORC2 (mTOR complex2) by mTOR, rictor (raptor independent), mLST8, mSIN1 (mammalian stress-activated protein kinase interacting protein 1), Protor-1 (Proteinobserved with rictor-1) is composed of five parts. The upstream and downstream effectors of mTORC1 and mTORC2 are almost identical, and they complement each other to coordinate cell cycle progression. The downstream effectors of mTORC1 mainly include ribosomal p70S6 kinase protein (S6kinase 1, S6K1) and eukaryotic initiation factor 4E binding protein 1 (4E binding protein 1, 4EBP1). mTORC1 affects the translation initiation of specific mRNA by regulating the phosphorylation of 4EBP1 and S6K1, thereby regulating protein synthesis and regulating cell growth and proliferation, and regulating protein synthesis is its main biological function. When mTORC1 is stimulated by factors such as growth factors, cell growth and proliferation can be promoted by activating downstream S6K1 (Thr389 site). Once mTORC1 is over-activated in the body, it will lead to overexpression of downstream 4EBP1 and S6K1, leading to uncontrolled cell proliferation, leading to excessive immunity or cancer. mTORC2 is primarily involved in the construction of cytoskeletal proteins, and mTORC2 up-regulates Akt levels by acting on the Ser473 site of Akt. (Tang Yu, a review of mTOR inhibitors [J]. Organic Chemistry, 2011, 31(7): 1144-1154.) The immunosuppressive and anticancer mechanisms of rapamycin are mainly through binding to the cellular receptor FKBP12. Inhibition of mTORC1, but inhibition of mTORC2 is extremely weak.

Rapamycin has strong anticancer activity, but it has two serious drawbacks: poor stability and poor water solubility. Rapamycin has a solubility in water of only 2.6 μg/ml and is almost insoluble in water. After years of research, it has been found that modifying the hydroxyl group at position 42 of rapamycin is a more reliable modification. The method of physicochemical properties of rapamycin has produced some marketed drugs based on this scheme.

The following are some of the 42 substituted rapamycin derivatives that have been marketed:

Temsirolimus, trade name Torisel, developed by Wyeth-Ayerst, is a 42-propionate derivative of rapamycin and is a rapamycin prodrug. It is the first FDA-approved anticancer drug for rapamycin derivatives (2007). Tansimus is a first-line treatment for patients with advanced renal cell carcinoma RCC (Real Cell carcinoma), which can extend the median survival of patients with early RCC by 3-6 months. Tansey Moss significantly inhibited T-cell proliferation, which is half maximal inhibitory concentration (IC ₅₀₎ is 0.8nmol / L, and with sunitinib (sunitinib) or sorafenib (sorafenib), etc. The combined use of kinase inhibitors .

Everolimus, trade name Zortress, developed by Novartis, is the first oral mTOR inhibitor with a chemical structure of 42-O-(2-hydroxyethyl)-rapamycin, which is superior in water solubility. Rapamycin is rapidly hydrolyzed when it enters the body. Experiments have shown that the oral bioavailability of everolimus is only 15%-30%, and the half-life in humans is 16-19h. First marketed in Sweden in 2003, initially used only as an immunosuppressive agent, it has now been approved for the treatment of RCC, pancreatic neuroendocrine tumors (PNET) and subependymal giant cell astrocytoma (SEGA). The FDA has approved everolimus oral tablets to prevent organ rejection in adult kidney transplant patients with low to moderate immune risk. Everolimus is used simultaneously with reduced amounts of cyclosporine A and basiliximab and corticosteroids. Approved Phase III trials have shown that everolimus prevents acute organ rejection and preserves kidney function.

Ridaforolimus (Ariad/Merck), modified at rapamycin C43, improved its solubility and PK value. Its research has completed Phase III clinical trials in patients with metastatic soft-tissue or bone sarcomas, with a 28% reduction in the risk of death in the medication group compared with the placebo group (NCT00538239). Ariad and Merck have submitted a new drug application for ridaforolimus to the FDA in 2012, but due to doubts about the efficacy of the FDA panel, the reporting process encountered some resistance.

Zotarolimus (Medtroni) approved the Zotarolimus Coated Coronary Stent System (Endeavor) in 2006. Compared with the bare metal coronary stent group, the applicator group can have Reduces arterial embolism, heart disease and heart infarction mortality in stent users.

Umiroliums (Biolimus, Biosensors) approved the launch of the Biolimus coated coronary stent system biolimus A9 in Europe in 2007. It has stronger immunosuppression and anti-inflammatory activity than rapamycin, which prevents cell migration and proliferation by arresting the cell cycle in G1, thereby preventing the occurrence of vascular restenosis.

Currently, there is a need to develop new rapamycin derivatives.

Summary of the invention

Through in-depth research and creative labor, the present invention has obtained a series of novel rapamycin carboxylate derivatives (compounds of formula I) having a quaternary ammonium salt structure, which not only greatly improved the rapamycin at Disadvantages in water solubility and metabolic properties (wherein the compound of Example 14 is 40,000 times more water soluble than rapamycin), and some of the compounds are superior to rapamycin in antitumor activity in vitro, and are less toxic to normal cells. Rapamycin has good drug-forming properties. The following invention is thus provided:

One aspect of the invention relates to a compound of formula I, a pharmaceutically acceptable salt thereof or a hydrate thereof:

among them,

R ₁ and R _{2 are} independently selected from H, A and B, and R ₁ and R _{2 are} not simultaneously H;

A=

B=

Where, in formula A or formula B,

The arrow indicates the site at which A or B is attached to the parent ring of formula I,

n is independently 1, 2, 3, 4, 5, 6 or 7;

R _{4 is} independently selected from the group consisting of fluorine, chlorine, bromine, iodine, nitro, cyano;

X ₁ , X ₂ , X ₃ , X ₄ , Y ₁ , Y ₂ , Y ₃ , Y ₄ , Y ₅ are each independently selected from C, S, O, N and Se atoms;

X ₁ to X ₂ , X ₂ to X ₃ , X ₃ to X ₄ , Y ₁ to Y ₂ , Y ₂ to Y ₃ , Y ₃ to Y ₄ , and Y ₄ to Y ₅ are each independently a single bond or a double bond. (X ₁ to X ₂ and Y ₁ to Y ₂ do not form a double bond, which is also known to those skilled in the art.)

Z ₁ , Z ₂ , Z ₃ , Z ₄ , Z ₅ , Z ₆ , Z ₇ , Z ₈ , Z _{9 are} independently selected from the group consisting of a hydrogen atom, a hydroxyl group, an aldehyde group, a carboxyl group, an amino group, a cyano group, a halogen, a C ₁ - C ₆ alkyl, C ₃ -C ₁₀ cycloalkyl, C ₁ -C ₆ alkoxy, C ₁ -C ₆ alkylthio, C ₃ -C ₁₀ cycloalkoxy, C ₁ -C ₆ alkenyl , alkynyl heterocycle, heterocycloalkyl, substituted heterocycloalkyl, aromatic ring, aromatic heterocyclic ring, benzoaromatic heterocyclic ring, wherein said C ₁ -C ₆ alkyl group, aromatic ring, aromatic heterocyclic ring, The benzoaromatic heterocyclic ring is unsubstituted or substituted by 1, 2, 3, 4 or 5 substituents independently selected from the group consisting of -F, -Cl, -Br, -I, nitro, hydroxy Amino, cyano, C ₁ -C ₆ alkylthio, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkenyl, C ₁ -C ₆ alkynyl and C ₁ -C ₆ alkoxy.

Specifically, the N atom forms a heterocyclic structure with X ₁ , X ₂ , X ₃ , X ₄ , or with Y ₁ , Y ₂ , Y ₃ , Y ₄ , Y ₅ ; specifically, a stable heterocyclic structure.

A compound of formula I according to any one of the invention, a pharmaceutically acceptable salt thereof or a hydrate thereof, wherein:

Z ₁ , Z ₂ , Z ₃ , Z ₄ , Z ₅ , Z ₆ , Z ₇ , Z ₈ , Z _{9 are} independently selected from the group consisting of a hydrogen atom, a hydroxyl group, an aldehyde group, a carboxyl group, an amino group, a cyano group, a halogen, a C ₁ - C ₃ alkyl, C ₃ -C ₆ cycloalkyl, C ₁ -C ₃ alkoxy, C ₁ -C ₃ alkylthio, C ₃ -C ₆ cycloalkoxy, C ₁ -C ₃ alkenyl .

A compound of formula I according to any one of the invention, a pharmaceutically acceptable salt thereof or a hydrate thereof, wherein:

In Formula A, the N atom and X ₁ , X ₂ , X ₃ , X ₄ form a thiazole ring, and/or

In the formula B, the N atom and Y ₁ , Y ₂ , Y ₃ , Y ₄ and Y ₅ constitute a pyridine ring.

A compound of formula I according to any one of the invention, a pharmaceutically acceptable salt thereof or a hydrate thereof, wherein:

Z ₁ , Z ₂ , Z ₃ , Z ₄ , Z ₅ , Z ₆ , Z ₇ , Z ₈ and Z _{9 are} independently selected from a hydrogen atom, a hydroxyl group and a methyl group.

A compound of formula I according to any one of the invention, a pharmaceutically acceptable salt thereof or a hydrate thereof, wherein:

R ₁ and R _{2 are} independently selected from H, carbonylmethyl-(4-methyl-thiazole R ₄ salt-3-yl), carbonylmethyl-(4,5-dimethyl-thiazole R ₄ salt-3 -yl), carbonylmethyl-(pyridine R ₄ salt-1-yl), carbonylmethyl-(3-hydroxy-pyridine R ₄ salt-1-yl), carbonylmethyl-(3-methyl-pyridine R ₄ salt-1-yl) and carbonylmethyl-(4-methyl-pyridine R ₄ salt-1-yl), wherein R _{4 is} independently selected from the group consisting of fluorine, chlorine, bromine, iodine, nitro and cyano;

And R ₁ and R _{2 are} not H at the same time.

A compound of formula I according to any one of the invention, a pharmaceutically acceptable salt thereof or a hydrate thereof, wherein:

R ₁ and R _{2 are} independently selected from H, carbonylmethyl-(4-methyl-thiazole bromide-3-yl), carbonylmethyl-(4,5-dimethyl-thiazolyl-3-yl ), carbonylmethyl-(pyridine bromide-1-yl), carbonylmethyl-(3-hydroxy-pyridine bromide-1-yl), carbonylmethyl-(3-methyl-pyridine bromide-1- And carbonylmethyl-(4-methyl-pyridyl bromide-1-yl);

And R ₁ and R _{2 are} not H at the same time.

In one embodiment of the invention, the compound shown in Table 1 below, a pharmaceutically acceptable salt thereof or a hydrate thereof is selected.

Table 1: Some compounds of the invention

A further aspect of the invention relates to a process for the preparation of a compound of formula I according to any of the preceding claims, which comprises the steps of any of the following methods (1) to (3):

Method (1): Preparation method of 31, 42-disubstituted compound of formula I

Method (2): Preparation of a 42-position monosubstituted compound of formula I

Method (3): Preparation of a monosubstituted compound of formula I at position 31

In the above methods (1)-(3),

R _{3 is} independently selected from the group consisting of F, Cl, Br, and I;

The meaning of the remaining symbols is independently as described in any of the preceding.

For the method (1), rapamycin is first reacted with an acid halide to form a diesterification product at 31 and 42 (intermediate 1), and then the product is reacted with a heterocyclic ring to form a 31,42 disubstituted quaternary ammonium salt. a rapamycin-like derivative.

For method (2), rapamycin is first reacted with a sufficient amount of tetramethylchlorosilane to form an intermediate. 2, then its 31-position ether bond hydrolyzes the intermediate 3 under the action of an acid, and then replaces the intermediate 4 of the intermediate with a halogenated acid halide, followed by acid to remove the tetramethylsilane at position 31. The ether affords the 42-monoesterified product (Intermediate 5); finally, the intermediate 5 is reacted with a nitrogen heterocycle to form a 42-position monosubstituted quaternary ammonium salt-like rapamycin derivative.

For the method (3), tert-butyldimethylsilyl chloride is substituted at position 42 of the intermediate 3 to give the intermediate 6; subsequently, the tetramethylsilyl ether at the position of 31 is hydrolyzed with an acid to obtain the intermediate 7; The acid halide is reacted with intermediate 7 to give intermediate 8; then tert-butyldimethylsilyl chloride is hydrolyzed under acidic conditions to give intermediate 9; finally the nitrogen heterocycle is reacted with intermediate 9 to give a 31-monosubstituted season. Ammonium salt rapamycin derivatives.

For the last step of the above method (1), that is, the reaction of the intermediate 1 to the final product of the method (1), a reaction reagent (herein referred to as R5 hereafter) may be added.

For the last step of the above method (2), that is, the reaction of the intermediate 5 of the method (2) to the final product, a reaction reagent (herein referred to as R5 hereafter) may be added.

For the last step of the above method (3), that is, the reaction of the intermediate 9 of the method (3) to the final product, a reaction reagent (herein referred to as R5 hereafter) may be added.

The above reaction reagent R5 is independently selected from a five-membered heterocyclic compound, a six-membered heterocyclic compound; specifically, one or more selected from the group consisting of thiazole, pyridine, and thiazole substituted by one or more C ₁ -C ₆ alkyl groups a C ₁ -C ₆ alkyl-substituted pyridine, a thiazole substituted with one or more halogen atoms, and a pyridine substituted with one or more halogen atoms; more specifically, selected from 4-methylthiazole, 4,5-di Methylthiazole, pyridine, 3-hydroxypyridine, 3-methylpyridine, and 4-methylpyridine.

A further aspect of the invention relates to a pharmaceutical composition comprising a compound according to any one of the invention, a pharmaceutically acceptable salt thereof or a hydrate thereof, and optionally a pharmaceutically acceptable excipient.

A series of compounds involved in the present invention will have excellent water solubility and are convenient to prepare into a variety of dosage forms. When administered orally, the compounds of the invention can be formulated into any orally acceptable formulation including, but not limited to, tablets, capsules, aqueous solutions or aqueous suspensions. Among them, the carrier used for the tablet generally includes lactose and corn starch, and a lubricant such as magnesium stearate may also be added. The diluent used in the capsule formulation generally comprises lactose and dried cornstarch. Aqueous suspension The formulations are usually prepared by mixing the active ingredient with suitable emulsifying and suspending agents. If desired, some sweeteners, fragrances or colorants may also be added to the above oral formulations.

The compounds of the present invention can also be administered in the form of a sterile injectable preparation, including sterile injectable aqueous or oily suspension or sterile injection solutions. Among them, carriers and solvents which can be used include water, Ringer's solution and isotonic sodium chloride solution.

A compound according to any one of the present invention, a pharmaceutically acceptable salt thereof or a hydrate thereof, or a pharmaceutical composition of the present invention, which is used as a medicament.

A compound according to any one of the present invention, a pharmaceutically acceptable salt thereof or a hydrate thereof, or a pharmaceutical composition of the present invention for use in inhibiting immunity, inhibiting mTOR, inhibiting mTORC1, inhibiting PI3K-Akt-mTOR signaling pathway, and inhibiting T lymphocyte proliferation, anti-tumor, promote tumor cell apoptosis, arrest cell cycle at G1, reduce arterial embolism, anti-aging, anti-Alzheimer's disease, prevent organ rejection, anti-inflammatory or antibacterial; specifically, Anti-tumor is the treatment and/or prevention and/or adjuvant treatment of renal cancer, lymphoma, lung cancer, liver cancer, breast cancer, neuroendocrine cancer, uterine sarcoma or gastric cancer.

The invention also relates to a method of treating and/or preventing and/or adjuvant treatment of kidney cancer, lymphoma, lung cancer, liver cancer, breast cancer, neuroendocrine cancer, uterine sarcoma or gastric cancer, comprising using an effective amount of any of the inventions A compound, a pharmaceutically acceptable salt thereof or a hydrate thereof thereof, or a step of the pharmaceutical composition of the present invention.

The dose of the compound of the present invention, a pharmaceutically acceptable salt thereof or a hydrate thereof, or the pharmaceutical composition of the present invention depends on many factors such as the nature and severity of the disease to be treated or adjuvanted, the sex of the patient or animal , age, body weight and individual response, the specific compound used, the route of administration and the number of doses. The above dosages may be administered in a single dosage form or divided into several, for example two, three or four dosage forms.

A further aspect of the invention relates to a compound according to any one of the invention, a pharmaceutically acceptable salt thereof or a hydrate thereof or a pharmaceutical composition of the invention for preparing an immunosuppressant, an mTOR inhibitor, an mTORC1 inhibitor, and a PI3K inhibitor -Akt-mTOR signaling pathway drugs, drugs that inhibit T lymphocyte proliferation, antitumor drugs, drugs that promote tumor cell apoptosis, drugs that arrest the cell cycle in G1, drugs that prevent organ rejection, and lower arterial emboli Use of a drug, an anti-aging drug, an anti-Alzheimer's disease drug, an anti-inflammatory drug or an antibacterial drug; in particular, the anti-tumor drug is a therapeutic and/or prophylactic and/or adjuvant treatment for renal cancer, lymphoma , lung cancer, liver cancer, breast cancer, neuroendocrine cancer, uterine sarcoma or gastric cancer drugs.

In a further aspect, the present invention relates to inhibiting immunity, inhibiting mTOR, inhibiting mTORC1, inhibiting PI3K-Akt-mTOR signaling pathway, inhibiting T lymphocyte proliferation, inhibiting tumor, promoting tumor cell apoptosis, and arresting cell cycle in vivo or in vitro. A method of reducing arterial embolism, anti-aging, anti-Alzheimer's disease, preventing organ rejection, anti-inflammatory or antibacterial, comprising using an effective amount of a compound according to any one of the present invention, a pharmaceutically acceptable salt thereof Or a hydrate thereof or a step of the pharmaceutical composition of the present invention.

In one embodiment of the invention, the above "in vitro" method is for non-therapeutic purposes.

A further aspect of the invention relates to a coronary stent comprising a pharmaceutical composition according to any one of the invention, a pharmaceutically acceptable salt thereof or a hydrate thereof or a pharmaceutical composition of the invention.

The invention further relates to the use of a compound according to any one of the invention, a pharmaceutically acceptable salt thereof or a hydrate thereof or a pharmaceutical composition of the invention for the preparation of a coronary stent as described above.

The invention further relates to a method of preparing a coronary stent as described above, comprising the step of using an effective amount of a compound according to any one of the invention, a pharmaceutically acceptable salt thereof or a hydrate thereof or a pharmaceutical composition of the invention.

In the present invention,

The term "C ₁ -C ₆ alkyl" refers to a straight or branched alkyl group having from 1 to 4 carbon atoms, such as methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, Tert-butyl, pentyl, 2-pentyl, isopentyl, neopentyl, hexyl, 2-hexyl, 3-hexyl, etc.; C ₁ -C ₄ alkyl, C ₁ -C ₃ alkyl or C ₁ - C ₂ alkyl groups can also be similarly understood. Particular alkyl groups are C ₁ -C ₄ alkyl, C ₁ -C ₃ alkyl or C ₁ -C ₂ alkyl.

The term "C ₁ -C ₆ alkoxy" refers to a straight or branched alkoxy group having from 1 to 6 carbon atoms, such as methoxy, ethoxy, propoxy, isopropoxy, n-butyl Oxyl, sec-butoxy, tert-butoxy, pentyloxy, 2-pentyloxy, isopentyloxy, neopentyloxy, hexyloxy, 2-hexyloxy, 3-hexyloxy, etc.; A similar understanding can also be made for C ₁ -C ₄ alkoxy, C ₁ -C ₃ alkoxy or C ₁ -C ₂ alkoxy. Particular alkoxy groups are C ₁ -C ₄ alkoxy, C ₁ -C ₃ alkoxy or C ₁ -C ₂ alkoxy.

The term "C ₁ -C ₆ alkylthio"" can be similarly understood as "C ₁ -C ₆ alkoxy", except that the oxygen atom is replaced by a sulfur atom.

The term "C ₃ -C ₁₀ cycloalkyl" refers to a saturated carbocyclic group having from 3 to 10 carbon atoms. The cycloalkyl group can be a monocyclic or polycyclic fused system and can be fused to the aromatic ring. Examples of such groups include cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl. The cycloalkyl group herein may be unsubstituted or substituted by various groups at one or more substitutable positions, as described in detail. For example, these cycloalkyl groups may be optionally substituted by C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, nitrile, halogen, hydroxy, amino, nitro, mono (C ₁ -C) ₆ ) alkylamino, bis(C ₁ -C ₆ )alkylamino, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C _} -C ₆ haloalkyl, C ₁ -C ₆ haloalkoxy . A similar understanding can also be made for the C ₃ -C ₆ cycloalkyl group.

The term "C ₃ -C ₁₀ cycloalkoxy" refers to a saturated carbocyclic alkoxy group having from 3 to 10 carbon atoms. The cycloalkoxy group may be a monocyclic or polycyclic fused system and may be fused to the aromatic ring. Examples of such groups include cyclopropoxy, cyclobutoxy, cyclopentyloxy and cyclohexyloxy. The cycloalkyl group herein may be unsubstituted or substituted by various groups at one or more substitutable positions, as described in detail. For example, these cycloalkoxy groups may be optionally substituted by C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, nitrile, halogen, hydroxy, amino, nitro, mono (C ₁ - C ₆ )alkylamino, di(C ₁ -C ₆ )alkylamino, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C _} -C ₆ haloalkyl, C ₁ -C ₆ haloalkoxy base. A similar understanding can also be made for the C ₃ -C ₆ cycloalkoxy group.

The term "C ₂ -C ₆ alkenyl" refers to an alkenyl group having 2 to 6 carbon atoms and at least one double bond, and includes ethenyl, propenyl, 1-but-3-enyl, 1-pent-3- - alkenyl group, 1-hexyl-5-enyl and the like; C ₃ -C ₅ alkenyl group also can be similarly understood. Preferred is a C ₃ -C ₅ alkenyl group.

The term "C ₂ -C ₆ alkynyl" refers to a hydrocarbon group having 2 to 6 carbon atoms and at least one triple bond, and includes ethynyl, propynyl, butynyl, pentyn-2-yl, and the like; C ₃ The -C ₅ alkynyl group can also be similarly understood. Preferred is a C ₃ -C ₅ alkynyl group.

The term "halogen" or "halogen atom" means a fluorine, chlorine, bromine, and iodine atom.

The term "aromatic ring" or "aryl" refers to having a single ring (such as phenyl), a polycyclic ring (such as biphenyl). Or a carbocyclic group of a plurality of fused rings (eg, 1,2,3,4-tetrahydronaphthyl, naphthyl) at least one of which is aromatic, such as halogen, lower alkane a mono-, di- or tri-substituted group of a lower alkoxy group, a trifluoromethyl group, an aryl group, a heteroaryl group and a hydroxy group.

The term "arylalkyl" refers to an alkyl group (as defined above) substituted with one or more aryl groups (as defined above). More preferred aryl alkyl is aryl -C _l -C ₃ alkyl. Examples include benzyl, phenylethyl and the like.

The term "aromatic heterocycle" or "heteroaryl" refers to one or more aromatic ring systems of a five-, six-, or seven-membered ring that includes a fused ring system of 5-10 atoms (at least one of which is It is aromatic) and the ring system contains at least one and up to four heteroatoms selected from nitrogen, oxygen or sulfur. Examples of heteroaryl groups are pyridyl, imidazolyl, pyrimidinyl, pyrazolyl, triazolyl, pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxazolyl, iso Thiazolyl, pyrrole ring, quinoline ring, isoquinoline ring, anthracene ring, benzimidazole, benzofuran ring, benzothiophene ring, benzothiazole ring, pyridazine ring and the like. It is optionally substituted mono, di or tri are, for example, halogen, lower alkyl, lower alkoxy, trifluoromethyl, aryl, heteroaryl and hydroxy.

The term "heterocycle" or "heterocyclyl" refers to one or more carbocyclic ring systems of a five-, six- or seven-membered ring comprising a fused ring system of from 4 to 10 atoms, said ring system containing At least one and up to four heteroatoms selected from nitrogen, oxygen or sulfur, provided that the ring of the group does not contain two adjacent O or S atoms. The fused ring system may be a heterocyclic ring fused to an aromatic group. Preferred heterocycles include, but are not limited to, pyrrolidinyl, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, piperidinyl, morpholine, cyclohexyl, piperazine, and the like, which may be substituted with the following groups: C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, nitrile, halogen, hydroxy, amino, nitro, mono(C ₁ -C ₆ )alkylamino, di(C ₁ -C ₆ ) alkane Amino, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C _} -C ₆ haloalkyl, C ₁ -C ₆ haloalkoxy.

The term "effective amount" when used in "in vivo" refers to a dose that can achieve a treatment, prevention, alleviation, and/or alleviation of a disease or condition described herein in a subject.

The term "subject" can refer to a patient or other animal that receives the composition of the invention to treat, prevent, ameliorate and/or alleviate the disease or condition of the invention, particularly a mammal, such as a human, a dog, a monkey, a cow, Horse and so on.

The term "disease and/or condition" refers to a physical state of the subject, the body The state is associated with the diseases and/or conditions described herein.

DRAWINGS

Figure 1: Inhibition of phosphorylation of the Thr389 site of S6K1 and the Ser473 site of Akt in tumor cell A549 by the compound of the example. A, Example Compounds 1-8; B, Example Compounds 9-17.

detailed description

The embodiments of the present invention will be described in detail below with reference to the accompanying drawings. Those who do not specify the specific conditions in the examples are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are conventional products that can be obtained commercially.

The melting point of the compound was determined by a SRY-1 type melting point apparatus, and the temperature was not corrected. The 1H-NMR spectrum was measured by a VARIAN INOVA 600 type nuclear magnetic apparatus; the mass spectrum was measured by an API-150EX LC/MS high resolution mass spectrometer.

Example 1: 31,42-O-carbonylmethyl-(4-methyl-thiazolyl-3-yl)-rapamycin Preparation of (Compound 1)

Step 1: 1 g (1.09 mmol) of rapamycin was dissolved in 10 ml of dry dichloromethane, cooled to -10 ° C, then 0.87 g (11 mmol) of dry pyridine was added. A solution of 2.2 g (10 mmol) of bromoacetyl bromide in 5 ml of dichloromethane was slowly added dropwise to the reaction mixture, and the mixture was added dropwise over 30 min. The pH of the reaction mixture was adjusted to neutral with 1 mol/L hydrochloric acid, washed with 20 ml×3 of distilled water, and then purified by column chromatography to obtain 0.73 g of white foamy product intermediate 1 (n=1, R ₃ =Br, R ₄ = Br).

Step 2: 0.73 g (0.63 mmol) of intermediate 1 (n=1, R ₃ =Br, R ₄ =Br) was dissolved in 20 ml of acetone, then added to the methyl 4-methylthiazole 0.94 g (9.45 mmol), and reacted at 60 ° C for 5 h. The column was separated to obtain 0.7 g of a pale yellow granular compound.

Mp 142-145 ° C; MS: 1193 [M-2Br-H]+, 597 [(M-2Br)/2]+; 1H-NMR (600 MHz, DMSO-d6 δ ppm), 10.18 (d, 2H), 8.07 ( s, 2H), 6.46 (s, 1H), 6.38 (m, 1H), 6.24 (m, 1H), 6.22 (m, 1H), 5.71 (s, 1H), 5.59 (s, 2H), 5.46 (m) , 1H), 5.33 (s, 1H), 4.91 (m, 3H), 4.53 (s, 2H).

Example 2: 42-O-carbonylmethyl-(4-methyl-thiazole bromide-3-yl)-rapamycin Preparation of compound 2)

Step 1: 2 g (2.19 mmol) of rapamycin was dissolved in 25 ml of dry ethyl acetate and cooled to 0-5 °C. To the reaction solution, 1.50 g (22 mmol) of imidazole was added, and a solution of 1.2 g (11 mmol) of trimethylchlorosilane in 5 ml of ethyl acetate was slowly added dropwise thereto for 30 minutes until completely converted into the intermediate 2. The mixture was slowly added dropwise to the reaction solution within 0.5 h. 10 ml of a mol/L H ₂ SO ₄ solution, after complete conversion of the intermediate 2, was extracted by column chromatography to obtain 1.3 g of a white foamy product intermediate 3.

Step 2: 1.3 g (1.3 mmol) of intermediate 3 was dissolved in 15 ml of dichloromethane, cooled to -10 ° C, then 1.03 g (13 mmol) dried pyridine. To the reaction liquid, a solution of 1.31 g (6.5 mmol) of bromoacetyl bromide in 5 ml was slowly added dropwise, and the mixture was added dropwise over 30 minutes, and the reaction was stopped after 10 min. The pH of the reaction mixture was adjusted to neutral with 1 mol/L hydrochloric acid, washed with 20 ml×3 of distilled water, and then purified by column chromatography to obtain 1.2 g of white foamy product intermediate 4 (n=1, R ₃ =Br, R ₄ = Br).

Step 3: 1.2 g (1.1 mmol) of intermediate 4 (n=1, R ₃ =Br, R ₄ =Br) was dissolved in 15 ml of acetone, and 1 ml/L of H ₂ SO ₄ aqueous solution 0.7 ml was added dropwise, 1 h plus After completion, the reaction was continued for 30 min and the reactants were completely converted. Column separation and purification, to give 1.05g of intermediate product as a white foam _{5 (n = 1, R 3} = Br, R 4 = Br) g.

Step 4: 1.05 g (0.95 mmol) of intermediate 5 (n = 1, R ₃ = Br, R ₄ = Br) was dissolved in 20 ml of acetone, and 0.94 g (9.5 mmol) of 4-methylthiazole was added, and reacted at 60 ° C for 5 h. The column was isolated to obtain 1.02 g of pale yellow granular compound 2.

Mp 130-133 ° C; MS: 1053.8 [M-Br] +; 1H-NMR (600 MHz, DMSO-d6 δ ppm), 10.16 (d, 1H), 8.06 (s, 1H), 6.45 (s, 1H), 6.38 ( m, 1H), 6.22 (m, 1H), 6.15 (m, 1H), 5.62 (s, 2H), 5.46 (M, 1H), 5.24 (s, 1H), 5.08 (d, 1H), 4.97 (s , 1H), 4.93 (s, 1H), 4.68 (m, 1H), 4.00 (s, 2H), 3.97 (s, 1H).

Example 3: 31-O-carbonylmethyl-(4-methyl-thiazolyl bromide-3-yl)-rapamycin Preparation of compound 3)

Step 1: 2 g (2 mmol) of intermediate 3 was dissolved in 20 mL of ethyl acetate and cooled to 0-5. 1.36 g (20 mmol) of imidazole was added to the reaction liquid, and 1.5 g (10 mmol) of t-butyldimethylsilyl chloride in 5 ml of ethyl acetate solution was slowly added dropwise over 30 min, and the reaction was continued for 30 min, and the column was separated to obtain 1.3 g of intermediate 6. 30 min. 0.5 ml of a 0.5 mol/L H ₂ SO ₄ solution was added dropwise to the inward reaction solution, and the reaction was completely converted after 1 h of addition. The column was separated and purified to give 1.05 g of white foamy product intermediate.

Step 2: 1.05 g (1.02 mmol) of intermediate 7 was dissolved in 10 mL dichloromethane, cooled to -10[deg.] C. then. A solution of 1.03 g (5.1 mmol) of bromoacetyl bromide in 5 ml was slowly added dropwise to the reaction mixture, and the mixture was added dropwise over 30 min. The reaction was stopped after continuing the reaction for 10 min. The pH of the reaction mixture was adjusted to neutral with 1 mol/L hydrochloric acid, washed with 20 ml×3 of distilled water, and then purified by column chromatography to obtain 0.73 g of white foamy product intermediate 8 (n=1, R ₃ =Br, R ₄ = Br).

Step 3: 0.73 g (0.64 mmol) of intermediate 8 (n=1, R ₃ =Br, R ₄ =Br) was dissolved in 10 ml of acetone, and 2 ml/L of H ₂ SO ₄ solution was added dropwise 0.5 ml, 30 min. After completion, the reaction was continued for 30 min and the reactants were completely converted. Column separation and purification gave 0.4 g of the white foamy product intermediate 9 (n = 1 , R ₃ =Br, R ₄ =Br).

Step 4: 0.4 g (0.39 mmol) of intermediate 9 (n = 1, R ₃ = Br, R ₄ = Br) was dissolved in 20 ml of acetone, 0.38 g (3.90 mmol) of 4-methylthiazole was added, and reacted at 60 ° C for 5 h. The column was isolated to give 0.27 g (0.24 mmol) of pale yellow granular compound 3.

Mp 130-132 ° C; MS: 1054 [M-Br]+; 1H-NMR (600 MHz, DMSO-d6 δ ppm), 10.16 (d, 1H), 8.05 (s, 1H), 6.45 (s, 1H), 6.37 ( m, 1H), 6.25 (m, 1H), 6.18 (m, 2H), 5.70 (s, 2H), 5.46 (m, 1H), 5.35 (s, 1H), 4.55 (d 2H), 4.04 (s, 1H).

Example 4: 31,42-O-carbonylmethyl-(4,5-dimethyl-thiazolyl bromide-3-yl)-ray Preparation of Papamycin (Compound 4)

For the preparation method, see Example 1 (n=1, R ₃ =Br, R ₄ =Br, R5=4,5-dimethylthiazole).

Mp 135-139 ° C; MS: 1220.9 [M-2Br-H]+; 1H-NMR (600 MHz, DMSO-d6 δ ppm), 10.03 (d, 2H), 6.46 (s, 1H), 6.37 (m, 1H), 6.24(m,1H), 6.18(m,1H), 6.12(m,1H), 5.76(m,3H), 5.62(s,2H), 5.46(m,1H),5.32(s,1H),5.32 (s, 1H), 4.93 (s, 1H), 4.83 (m, 2H), 4.61 (s, 1H), 4.55 (s, 1H), 4.52 (s, 1H).

Example 5: 42-O-carbonylmethyl-(4,5-dimethyl-thiazolyl-3-yl)-rapamycin Preparation of compound (compound 5)

For the preparation method, see Example 2 (n=1, R ₃ =Br, R ₄ =Br, R5=4,5-dimethylthiazole).

Mp 135-138 ° C; MS: 1067.9 [M-Br]+; 1H-NMR (600 MHz, DMSO-d6 δ ppm), 10.03 (d, 1H), 6.49 (s, 1H), 6.37 (m, 1H), 6.21. m,1H), 6.14 (m, 2H), 5.62 (m, 2H), 5.46 (m, 1H), 5.23 (s, 1H), 5.08 (s, 1H), 4.97 (s, 1H), 4.93 (s , 1H), 4.68 (s, 1H), 4.06 (s, 2H), 4.01 (s, 1H).

Example 6: 31-O-carbonylmethyl-(4,5-dimethyl-thiazolyl-3-yl)-rapamycin Preparation of compound (compound 6)

For the preparation method, see Example 3 (n=1, R ₃ =Br, R ₄ =Br, R5=4,5-dimethylthiazole).

Mp 125-127 ° C; MS: 1067.7 [M-Br]+; 1H-NMR (600 MHz, DMSO-d6 δ ppm), 10.02 (d, 1H), 6.45 (s, 1H), 6.37 (m, 1H), 6.25 ( m, 1H), 6.20 (m, H), 6.15 (m, H), 5.70 (m, 2H), 5.45 (m, 1H), 5.33 (s, 1H), 5.08 (s, 1H), 4.93 (s) , 1H), 4.81 (m, 1H), 4.56 (m, 3H), 4.03 (m, 1H).

Example 7: 31,42-O-carbonylmethyl-(pyridine bromide-1-yl)-rapamycin (combination Preparation of substance 7)

For the preparation method, see Example 1 (n = 1, R ₃ = Br, R ₄ = Br, R5 = pyridine).

Mp 140-142 ° C; MS: 1153 [M-Br]+; 1H-NMR (600 MHz, DMSO-d6 δ ppm), 8.96 (d, 4H), 8.72 (m, 2H), 8.22 (m, 4H), 6.44 ( s, 1H), 6.40 (m, 1H), 6.24 (m, 1H), 6.17 (m, 2H), 5.87 (m, 1H), 5.79 (m, 1H), 5.48 (m, 1H), 5.29 (s) , 1H), 4.95 (m, 2H), 4.81 (s, 1H), 4.52 (m, 3H).

Example 8: 31,42-O-carbonylmethyl-(3-hydroxy-pyridyl bromide-1-yl)-rapamycin Preparation of (Compound 8)

For the synthesis method, see Example 1 (n = 1, R ₃ = Br, R ₄ = Br, R 5 = 3-hydroxypyridine).

Mp 120-124 ° C; MS: 1185.4 [M-2Br-H]+; 1H-NMR (600 MHz, DMSO-d6 δ ppm), 12.04 (s, 2H), 8.60 (m, 4H), 8.00 (m, 4H), 6.43 (m, 2H), 6.12 (m, 3H), 5.61 (m, 3H), 5.56 (m, 1H).

Example 9: 31,42-O-carbonylmethyl-(3-methyl-pyridyl bromide-1-yl)-rapamycin Preparation of (Compound 9)

For the preparation method, see Example 1 (n=1, R ₃ =Br, R ₄ =Br, R5=3-methylpyridine).

Mp 159-162 ° C; MS: 1181.2 [M-2Br-H]+; 1H-NMR (600 MHz, DMSO-d6 δ ppm), 9.00 (s, 1H), 8.89 (m, 2H), 8.81 (m, 1H), 8.57 (s, 2H), 8.14 (m, 2H), 6.46 (s, 1H), 6.38 (m, 1H), 6.23 (m, 1H), 6.14 (m, 2H), 5.80 (m, 5H), 5.46 (m, 1H), 5.29 (m, 1H).

Example 10: 42-O-carbonylmethyl-(pyridine bromide-1-yl)-rapamycin (compound) Preparation of 10)

For the preparation method, see Example 2 (n = 1, R ₃ = Br, R ₄ = Br, R5 = pyridine).

Mp 135-137 ° C; MS: 1034.1 [M-Br]+; 1H-NMR (600 MHz, DMSO-d6 δ ppm), 9.07 (d, 1H), 9.02 (m, 1H), 8.71 (m, 1H), 8.24 m, 2H), 8.14 (m, 2H), 6.45 (s, 1H), 6.41 (m, 1H), 6.22 (m, 1H), 6.14 (m, 1H), 5.80 (m, 5H), 5.67 (m) , 2H), 5.44 (m, 1H), 5.25 (s, 1H), 5.08 (m, 1H).

Example 11: 42-O-carbonylmethyl-(3-methyl-pyridyl bromide-1-yl)-rapamycin Preparation of compound 11)

For the preparation method, see Example 2 (n = 1, R ₃ = Br, R ₄ = Br, R 5 = 3-methylpyridine).

Mp 138-140 ° C; MS: 1048 [M-Br]+; 1H-NMR (600 MHz, DMSO-d6 δ ppm), 9.07 (d, 1H), 9.02 (m, 1H), 8.71 (m, 1H), 8.24 ( m, 2H), 8.14 (m, 2H), 6.45 (s, 1H), 6.41 (m, 1H), 6.22 (m, 1H), 6.14 (m, 1H), 5.80 (m, 5H), 5.67 (m) , 2H), 5.44 (m, 1H), 5.25 (s, 1H), 5.08 (m, 1H).

Example 12: 42-O-carbonylmethyl-(3-hydroxy-pyridyl bromide-1-yl)-rapamycin Preparation of compound 12)

Preparation see Example _{2 (n = 1, R 3} = Br, R 4 = Br, R5 = 3- hydroxypyridine).

Mp 146-148 ° C; MS: 1050 [M-Br]+; 1H-NMR (600 MHz, DMSO-d6 δ ppm), 12.08 (s, 1H), 8.60 (m, 1H), 8.53 (m, 1H), 8. m, 2H), 6.45 (m, 2H), 6.38 (s, 1H), 6.22 (m, 1H), 6.12 (m, 2H), 5.60 (m, 2H), 5.48 (m, 1H), 5.25 (m) , 1H), 5.08 (m, 1H).

Example 13: 42-O-carbonylmethyl-(4-methyl-pyridyl bromide-1-yl)-rapamycin Preparation of compound 13)

For the preparation method, see Example 2 (n=1, R ₃ =Br, R ₄ =Br, R5=4-methylpyridine).

Mp 157-160 ° C; MS: 1048.1 [M-Br]+; 1H-NMR (600 MHz, DMSO-d6 δ ppm), 8.89 (d, 1H), 8.05 (m, 2H), 6.45 (s, 1H), 6.41 ( m, 1H), 6.22 (m, 1H), 6.12 (m, 2H), 5.61 (m, 3H), 5.46 (m, 1H), 5.25 (m, 1H), 5.08 (d, 1H), 4.93 (m) , 2H).

Example 14: 31,42-O-carbonylmethyl-(4-methyl-pyridyl bromide-1-yl)-rapamycin Preparation of compound (compound 14)

For the preparation method, see Example 1 (n=1, R ₃ =Br, R ₄ =Br, R5=4-methylpyridine).

Mp 181-183 ° C; MS: 1181.2 [M-2Br-H]+; 1H-NMR (600 MHz, DMSO-d6 δ ppm), 8.90 (d, 2H), 8.78 (d, 2H), 8.06 (m, 4H), 6.46(d,1H), 6.38(m,1H), 6.20(m,1H), 6.14(m,2H), 5.80(d,1H), 5.62(m,4H),5.47(m,1H),5.29 (s, 1H).

Example 15: 31-O-carbonylmethyl-(3-methyl-pyridyl bromide-1-yl)-rapamycin Preparation of compound 15)

For the preparation method, see Example 3 (n = 1, R ₃ = Br, R ₄ = Br, R 5 = 3-methylpyridine).

Mp 138-140 ° C; MS: 1048.1 [M-Br]+; 1H-NMR (600 MHz, DMSO-d6 δ ppm), 8.88 (d, 1H), 8.80 (d, 1H), 8.57 (m, 1H), 8.12 ( m,1H), 6.45 (m, 1H), 6.40 (m, 1H), 6.23 (m, 1H), 6.17 (m, 2H), 5.81 (d, 1H), 5.74 (m, 2H), 5.47 (m) , 1H) 5.28 (s, 1H).

Example 16: 31-O-carbonylmethyl-(pyridine bromide-1-yl)-rapamycin (compound) Preparation of 16)

For the preparation method, see Example 3 (n = 1, R ₃ = Br, R ₄ = Br, R 5 = pyridine).

Mp 140-142 ° C; MS: 1034 [M-Br]+; 1H-NMR (600 MHz, DMSO-d6 δ ppm), 8.96 (d, 2H), 8.72 (m, 1H), 8.22 (m, 2H), 6.44 ( s, 1H), 6.40 (m, 1H), 6.24 (m, 1H), 6.17 (m, 2H), 5.87 (m, 1H), 5.79 (m, 1H), 5.48 (m, 1H), 5.29 (s) , 1H), 4.95 (m, 2H), 4.81 (s, 1H), 4.52 (m, 3H).

Example 17: 31-O-carbonylmethyl-(4-methyl-pyridyl bromide-1-yl)-rapamycin Preparation of compound 17)

For preparation see Example _{3 (n = 1, R 3} = Br, R 4 = Br, R5 = 4- methylpyridine).

Mp 145-148 ° C; MS: 1048.1 [M-Br]+; 1H-NMR (600 MHz, DMSO-d6 δ ppm), 8.77 (d, 2H), 8.03 (d, 2H), 6.44 (s, 1H), 6.40 ( m, 1H), 6.23 (m, 1H), 6.16 (m, 2H), 5.80 (d, 1H), 5.70 (d, 1H), 5.44 (m, 1H), 5.29 (m, 1H), 5.29 (s) , 1H).

Example 18: Solubility test

Taking the compounds 1-17 of the present invention, respectively, according to the solubility test method described in the second part of the Chinese Pharmacopoeia 2010 edition, the results are shown in Table 2:

Table 2: Water solubility list of compounds 1-17

As can be seen from the data in Table 2, the water solubility of the compounds of the present invention is significantly higher than that of rapamycin, and some compounds are even higher by three to four orders of magnitude.

Example 19: Evaluation of rat primary hepatocyte toxicity

Experimental method: The liver leaves of SD rats were placed in a disinfection plate, and an appropriate amount of liver cell washing solution (pre-cooling at 4 ° C) was added to shred the liver and remove fibrous connective tissue to prepare a mixed liver cell suspension, which was filtered through a 200-mesh sieve. Into a 50ml centrifuge tube, centrifuge at 500rpm for 1-2 minutes; remove the supernatant, add RPMI 1640 (4 ° C pre-cooling) 20-30ml wash 3 times; take an equal volume of liver cell suspension suspended in Percoll separation solution I, invert and mix After centrifugation at 800 rpm for 10 min at 4 ° C, the supernatant was discarded and the liver cells were washed once with PBS (800 rpm, 5 min). The hepatocyte pellet was added to the hepatocyte culture medium and resuspended to a volume of 10 ml, and the appropriate amount was counted and the survival rate was judged by 0.4% trypan blue staining. Hepatocytes with a viability of more than 90% were seeded at a density of 2x103 in 96-well plates, 100 μl of RPMI 1640 medium was added to each well, and cultured at 37 ° C, 5% CO ₂ for 24 h. The compound was diluted to the desired concentration with DMSO, 1 μl of each well was added (the final concentration of the compound was usually 1000 μM, 10× gradient dilution, 6 gradients), and the blank was added to 1 μl of DMSO.

After the cells were cultured at 37 ° C, 5% CO _{2 for} 24 h, 100 μl of ATP detection reagent was added and incubated for 15 minutes with shaking. The fluorescence values of each well were read on a microplate reader. The cell viability after treatment of the compounds of each example was calculated, and the GI ₅₀ value (concentration of the compound added when 50% of cell growth was inhibited) was calculated. The results are shown in Table 4. The toxicity of the compound is significantly less than that of rapamycin.

Table 4: GI ₅₀ values of some example compound concentrations on rat primary hepatocytes

Compound	GI 50 (μM)
		Rapamycin	24.822
Example 11 compound	71.753
		Example 13 compound	44.936
Example 17 compound	54.311

As can be seen from the data in Table 4, the compounds of the present invention are significantly less toxic than rapamycin.

Example 20: Inhibitory activity of compounds against mTORC1 and mTORC2

The molecular mechanism of action of the compound of the example in tumor cell A549 was evaluated.

Experimental materials: DMEM high glucose cell culture medium (Hyclone), fetal bovine serum (FBS) (Gibco), penicillin, streptomycin purchased from North China Pharmaceutical Co., Ltd., phosphate physiological saline buffer (PBS) purchased from Gibco Trypsin and dimethyl sulfoxide (DMSO) are products of Sigma. The A549 cell line (human lung adenocarcinoma cell line) was purchased from ATCC. Mouse anti-human p-p70S6K (T389) monoclonal antibody was purchased from Cell Signaling Technology, mouse anti-human p70S6K monoclonal antibody was purchased from Cell Signaling Technology, and mouse anti-human p-AKT (S473) monoclonal antibody was purchased from Cell Signaling Technology The company, mouse anti-human AKT monoclonal antibody was purchased from Cell Signaling Technology. Horseradish peroxidase-labeled goat anti-mouse and goat anti-mouse monoclonal antibodies were purchased from Cell Signaling Technology. The cell lysate was purchased from Beijing Suo Laibao Technology Co., Ltd., the loading buffer was purchased from Biyuntian Company, the SDS electrophoresis buffer was purchased from Biyuntian Company, the membrane buffer was purchased from Biyuntian Company, and the TBS was purchased from Biyuntian Company. TBST was purchased from Biyuntian Company. ECL chemiluminescence solution was purchased from Beijing Pulilai Gene Technology Co., Ltd., developer and fixer were purchased from Shijiazhuang Chemical Industry No. 10 Factory, NC film was purchased from Whatman Company, film was purchased from Ruiyi (Xiamen) Medical Equipment Co., Ltd., skim milk powder Purchased from Beijing Xikai Innovation Technology Co., Ltd.

experimental method:

A549 cells were plated into 96-well plates. When the cells were grown to a density of 80%-90%, they were serum-free and starved overnight. The next day, 167 nM insulin and compound were incubated for 2 hours, and then the lysed cells were detected by Western blotting. The phosphorylation levels of the compound at the Thr389 site of S6K1 (corresponding to mTORC1) and the Ser473 site of Akt (corresponding to mTORC2) were used to semi-quantitatively reflect the inhibitory levels of mTORC1 and mTORC2 by the compounds (results shown) In the middle, no blot indicates that the expression of the corresponding protein was inhibited, and there was a blot indicating that it was not inhibited). The positive control compound was selected for Rapamycin. The evaluation results are shown in Figure 1 (Figure 1A and Figure 1B). Specific steps are as follows:

1. Treat cells:

1) Inoculate the cells into a six-well plate, and after 24 hours, the cells are covered with 80%-90%;

2) After washing the cells once with PBS, exchange 2 mL of serum-free medium and starve overnight;

3) 2 μL of the cell culture medium containing the test compound (20 μM) and insulin (167 nM) was added to the treated cells for 2 h.

2. Lysis cells:

1) The cells in the 6-well plate were blown down with 10 ml of PBS, and centrifuged at 1500 rpm for 5 min;

2) After discarding the supernatant, add 60 μl of cell lysis buffer (containing protease inhibitor), cleave on ice for 20-30 min, shake it with an oscillator for 2-3 s after 10 min, and put it back on ice;

3) shaking for 3 s before centrifugation, then centrifuging at 4 ° C, 13000 rpm for 10 min;

4) Add 2μ loading buffer 60μl, boil water bath for 5min, -20°C or -80°C for storage;

3. Western blotting experiments:

1) SDS-PAGE, first run to the concentrated glue with 80V volts, then run the separation gel with 120V voltage;

2) Transfer film, wet transfer, in ice bath, 250mA, 150min;

3) 5% milk is blocked at room temperature for 1-2 hours (can be overnight at 4 ° C);

4) Add primary antibody (diluted with TBS), overnight at 4 °C;

5) TBST was washed 3 x 10 min.

6) Add a 1:2000 diluted secondary antibody (diluted with 3% skim milk powder, TBS configuration), and incubate on the parafilm for 2 h at room temperature;

7) TBST was washed 3 x 10 min.

8) ECL chemiluminescence coloring solution A, B solution 500μl each mixed in a dish, rinsed for 5min;

9) placing the film in the exposure clip, exposing it to the dark room for a certain period of time, then placing it in the developer for 2 min, fixing the solution for 5 min, washing the film with tap water, and drying;

10) Save the photo.

The results indicate that the compounds of the present invention are effective in inhibiting mTORC1, and the inhibitory effect is not weaker than that of rapamycin (rapamycin also inhibits only mTORC1, so our compound is not weaker than rapamycin).

Example 21: Tumor cell inhibition assay

The activity of the example compounds against tumor cell A549 was evaluated.

Experimental materials: DMEM high glucose cell culture medium (Hyclone), fetal bovine serum (FBS) (Gibco), penicillin, streptomycin purchased from North China Pharmaceutical Co., Ltd., phosphate physiological saline buffer (PBS) purchased from Gibco , Cell

Cell viability assay reagents were purchased from Promega, trypsin and dimethyl sulfoxide (DMSO) for Sigma. The A549 cell line (human lung adenocarcinoma cell line) was purchased from ATCC.

experimental method:

A white-walled 96-well plate (Costar) was inoculated at a number of 5000 cells per well, and cultured at 37 ° C under 5% CO ₂ for 24 hours. The test compound was dissolved to 100 mM using DMSO as a mother liquor of the compound.

The compound was diluted with a DMEM medium containing 2% FBS at a concentration gradient of 3 and a concentration ranging from 100 μM to 3 nM. Each dilution compound was added to the cultured 96-well plate cells at 100 μl per well. The cells were cultured for 72 h under 37 ° C CO ₂ conditions, and the supernatant was discarded, and the cell viability assay was performed.

Will Cell

The reaction buffer and the substrate were mixed in equal proportions and then added to a 96-well plate at 100 μl per well. The cells were shaken horizontally for 4 min to induce cell lysis. The reaction was equilibrated for 15 min at room temperature to stabilize the reaction signal. Chemiluminescence detectors were used to detect the chemiluminescence units of each well in a 96-well plate.

The inhibition rate of each dilution of each compound was calculated according to the chemiluminescence detection value of each well, and the different gradients of each compound were subjected to S-shaped curve fitting using Origin 8.0 software to calculate the EC ₅₀ value. The results are shown in Table 5.

Table 5: Example Compound concentration portion ₅₀ tumor cell line A549 values EC

Compound	IC 50 (μM)
		Rapamycin	49.35
Example 1 compound	17.39
		Example 2 compound	19.31
Example 3 compound	3.04

Example 4 compound	6.23
		Example 5 compound	7.40
Example 6 compound	52.26
		Example 7 compound	46.68
Example 8 compound	45.98
		Example 9 compound	25.12
Example 10 compound	66.32
		Example 11 compound	52.80
Example 12 compound	51.48
		Example 13 compound	44.80
Example 14 compound	44.0
		Example 15 compound	52.70
Example 16 compound	57.71
		Example 17 compound	40.80

The results indicate that the compounds of the present invention are effective in tumor cells, and some compounds are even superior to rapamycin, and have potential for the preparation of antitumor drugs.

Example 22: Tumor cell resistant strain inhibition experiment

The activity of the compound of the example against the tumor cell multidrug resistant strain MES-SA was evaluated.

Experimental materials: DMEM high glucose cell culture medium (Hyclone), fetal bovine serum (FBS) (Gibco), penicillin, streptomycin purchased from North China Pharmaceutical Co., Ltd., phosphate physiological saline buffer (PBS) purchased from Gibco , Cell

Cell viability assay reagents were purchased from Promega, trypsin and dimethyl sulfoxide (DMSO) for Sigma. The MES-SA cell line (human uterine sarcoma cell line) was purchased from ATCC. MRC5 cells (human embryonic lung fibroblasts) were purchased from ATCC.

experimental method:

A white-walled 96-well plate (Costar) was inoculated at a number of 5000 cells per well, and cultured at 37 ° C under 5% CO ₂ for 24 hours. The test compound was dissolved to 100 mM using DMSO as a mother liquor of the compound.

The compound was diluted with a DMEM medium containing 2% FBS at a concentration gradient of 3 and a concentration ranging from 100 μM to 0.046 μM. Each dilution compound was added to the cultured 96-well plate cells at 100 μl per well. The cells were cultured for 72 h under 37 ° C CO ₂ conditions, and the supernatant was discarded, and the cell viability assay was performed.

Will Cell

The reaction buffer and the substrate were mixed in equal proportions and then added to a 96-well plate at 100 μl per well. The cells were shaken horizontally for 4 min to induce cell lysis. The reaction was equilibrated for 15 min at room temperature to stabilize the reaction signal. Chemiluminescence detectors were used to detect the chemiluminescence units of each well in a 96-well plate.

The inhibition rate of each dilution of each compound was calculated according to the chemiluminescence detection value of each well, and the different gradients of each compound were subjected to S-shaped curve fitting using Origin 8.0 software to calculate the EC ₅₀ value. The ratio of the test compound and ₅₀ values EC MRC5 cells ₅₀ values EC MES-SA cells is the therapeutic index of the compound. The experimental results are shown in Table 6 below.

Table 6: Examples for tumor cells Compound EC ₅₀ values of MES-SA and the therapeutic index portions embodiment

Compound	IC 50 (μM)	Treatment index
			Rapamycin	<0.046	>635.09
Example 1 compound	<0.046	>259.8
			Example 2 compound	<0.046	>1811.9
Example 3 compound	<0.046	>1501.1
			Example 4 compound	<0.046	>787.2
Example 5 compound	<0.046	>1371.1
			Example 6 compound	<0.046	>455.1

Example 7 compound	<0.046	>442.7
			Example 8 compound	<0.046	>1441.6
Example 9 compound	7.21	12.1
			Example 10 compound	10.93	3.4
Example 11 compound	<0.046	>1888.7
			Example 12 compound	<0.046	>146.5
Example 13 compound	<0.046	>1559.0
			Example 14 compound	<0.046	>2136.8
Example 15 compound	<0.046	>331.5
			Example 16 compound	<0.046	>364.8
Example 17 compound	<0.046	>317.7

The results showed that the compound of the present invention can effectively inhibit the proliferation of the multi-drug resistant strain MES-SA of tumor cells, and its inhibitory activity against normal cells is generally lower than that of MES-SA, and has a good therapeutic index and safety. Good sex. Some compounds have better efficacy than rapamycin and have potential for use in the preparation or as an anti-tumor drug.

While the present invention has been described in detail, it will be understood by those skilled in the art . The full scope of the invention is given by the appended claims and any equivalents thereof.

Claims (14)

1. a compound of formula I, a pharmaceutically acceptable salt thereof or a hydrate thereof:

   

   among them,

   R ₁ and R _{2 are} independently selected from H, A and B, and R ₁ and R _{2 are} not simultaneously H;

   

   Where, in formula A or formula B,

   The arrow indicates the site at which A or B is attached to the parent ring of formula I;

   n is independently 1, 2, 3, 4, 5, 6 or 7;

   R _{4 is} independently selected from the group consisting of fluorine, chlorine, bromine, iodine, nitro, cyano;

   X ₁ , X ₂ , X ₃ , X ₄ , Y ₁ , Y ₂ , Y ₃ , Y ₄ , Y ₅ are each independently selected from C, S, O, N and Se atoms;

   X ₁ to X ₂ , X ₂ to X ₃ , X ₃ to X ₄ , Y ₁ to Y ₂ , Y ₂ to Y ₃ , Y ₃ to Y ₄ , and Y ₄ to Y ₅ are each independently a single bond or a double bond. ;

   Z ₁ , Z ₂ , Z ₃ , Z ₄ , Z ₅ , Z ₆ , Z ₇ , Z ₈ , Z _{9 are} independently selected from the group consisting of a hydrogen atom, a hydroxyl group, an aldehyde group, a carboxyl group, an amino group, a cyano group, a halogen, a C ₁ - C ₆ alkyl, C ₃ -C ₁₀ cycloalkyl, C ₁ -C ₆ alkoxy, C ₁ -C ₆ alkylthio, C ₃ -C ₁₀ cycloalkoxy, C ₁ -C ₆ alkenyl , alkynyl heterocycle, heterocycloalkyl, substituted heterocycloalkyl, aromatic ring, aromatic heterocyclic ring, benzoaromatic heterocyclic ring, wherein said C ₁ -C ₆ alkyl group, aromatic ring, aromatic heterocyclic ring, The benzoaromatic heterocyclic ring is unsubstituted or substituted by 1, 2, 3, 4 or 5 substituents independently selected from the group consisting of -F, -Cl, -Br, -I, nitro, hydroxy Amino, cyano, C ₁ -C ₆ alkylthio, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkenyl, C ₁ -C ₆ alkynyl and C ₁ -C ₆ alkoxy.
2. A compound of formula I according to claim 1, a pharmaceutically acceptable salt thereof or a hydrate thereof, wherein:

   Z ₁ , Z ₂ , Z ₃ , Z ₄ , Z ₅ , Z ₆ , Z ₇ , Z ₈ , Z _{9 are} independently selected from the group consisting of a hydrogen atom, a hydroxyl group, an aldehyde group, a carboxyl group, an amino group, a cyano group, a halogen, a C ₁ - C ₃ alkyl, C ₃ -C ₆ cycloalkyl, C ₁ -C ₃ alkoxy, C ₁ -C ₃ alkylthio, C ₃ -C ₆ cycloalkoxy, C ₁ -C ₃ alkenyl .
3. A compound of the formula I according to claim 1 or 2, a pharmaceutically acceptable salt thereof or a hydrate thereof, which satisfies any one or more of the following 1) to 3):

   1) In the formula A, the N atom and X ₁ , X ₂ , X ₃ , X ₄ constitute a thiazole ring;

   2) In the formula B, the N atom and Y ₁ , Y ₂ , Y ₃ , Y ₄ , Y ₅ constitute a pyridine ring;

   3) Z ₁ , Z ₂ , Z ₃ , Z ₄ , Z ₅ , Z ₆ , Z ₇ , Z ₈ and Z _{9 are} independently selected from a hydrogen atom, a hydroxyl group and a methyl group.
4. A compound of formula I according to claim 1, a pharmaceutically acceptable salt thereof or a hydrate thereof, wherein:

   R ₁ and R _{2 are} independently selected from H, carbonylmethyl-(4-methyl-thiazole R ₄ salt-3-yl), carbonylmethyl-(4,5-dimethyl-thiazole R ₄ salt-3 -yl), carbonylmethyl-(pyridine R ₄ salt-1-yl), carbonylmethyl-(3-hydroxy-pyridine R ₄ salt-1-yl), carbonylmethyl-(3-methyl-pyridine R ₄ salt-1-yl) and carbonylmethyl-(4-methyl-pyridine R ₄ salt-1-yl), wherein R _{4 is} independently selected from the group consisting of fluorine, chlorine, bromine, iodine, nitro and cyano, and R ₁ , R _{2 are} not H at the same time; or

   R ₁ and R _{2 are} independently selected from H, carbonylmethyl-(4-methyl-thiazole bromide-3-yl), carbonylmethyl-(4,5-dimethyl-thiazolyl-3-yl ), carbonylmethyl-(pyridine bromide-1-yl), carbonylmethyl-(3-hydroxy-pyridine bromide-1-yl), carbonylmethyl-(3-methyl-pyridine bromide-1- And carbonylmethyl-(4-methyl-pyridyl bromide-1-yl), and R ₁ and R _{2 are} not H at the same time.
5. A compound of the formula I according to claim 1, a pharmaceutically acceptable salt thereof or a hydrate thereof, wherein the compound is selected from the group consisting of:

   31,42-O-carbonylmethyl-(4-methyl-thiazolyl-3-yl)-rapamycin,

   42-O-carbonylmethyl-(4-methyl-thiazolyl-3-yl)-rapamycin,

   31-O-carbonylmethyl-(4-methyl-thiazolyl-3-yl)-rapamycin,

   31,42-O-carbonylmethyl-(4,5-dimethyl-thiazolyl-3-yl)-rapamycin,

   42-O-carbonylmethyl-(4,5-dimethyl-thiazolyl-3-yl)-rapamycin,

   31-O-carbonylmethyl-(4,5-dimethyl-thiazolyl-3-yl)-rapamycin,

   31,42-O-carbonylmethyl-(pyridyl bromide-1-yl)-rapamycin,

   31,42-O-carbonylmethyl-(3-hydroxy-pyridyl bromide-1-yl)-rapamycin,

   31,42-O-carbonylmethyl-(3-methyl-pyridyl bromide-1-yl)-rapamycin,

   42-O-carbonylmethyl-(pyridyl bromide-1-yl)-rapamycin,

   42-O-carbonylmethyl-(3-methyl-pyridyl bromide-1-yl)-rapamycin,

   42-O-carbonylmethyl-(3-hydroxy-pyridyl bromide-1-yl)-rapamycin,

   42-O-carbonylmethyl-(4-methyl-pyridyl bromide-1-yl)-rapamycin,

   31,42-O-carbonylmethyl-(4-methyl-pyridyl bromide-1-yl)-rapamycin,

   31-O-carbonylmethyl-(3-methyl-pyridyl bromide-1-yl)-rapamycin,

   31-O-carbonylmethyl-(pyridyl bromide-1-yl)-rapamycin, and

   31-O-carbonylmethyl-(4-methyl-pyridyl bromide-1-yl)-rapamycin.
6. A process for the preparation of a compound of formula I according to any one of claims 1 to 5, which comprises the steps of any of the following methods (1) to (3):

   Method (1): Preparation method of 31, 42-disubstituted compound of formula I

   

   Method (2): Preparation of a 42-position monosubstituted compound of formula I

   

   Method (3): Preparation of a monosubstituted compound of formula I at position 31

   

   In the above methods (1)-(3),

   R _{3 is} independently selected from the group consisting of F, Cl, Br, and I;

   The meaning of the remaining symbols is independently as set forth in any one of claims 1 to 5.
7. The preparation method according to claim 6, wherein in the last step of the method (1), the method (2) or the method (3), a reaction reagent is added, and the reaction reagent is independently selected from a five-membered heterocyclic ring. a compound, a six-membered heterocyclic compound; specifically, a pyridine selected from the group consisting of thiazole, pyridine, thiazole substituted by one or more C ₁ -C ₆ alkyl groups, substituted by one or more C ₁ -C ₆ alkyl groups, a thiazole substituted with a plurality of halogen atoms and a pyridine substituted with one or more halogen atoms; more specifically, selected from the group consisting of 4-methylthiazole, 4,5-dimethylthiazole, pyridine, 3-hydroxypyridine, 3- Methylpyridine and 4-methylpyridine
8. A pharmaceutical composition comprising the compound of any one of claims 1 to 5, a pharmaceutically acceptable salt thereof, or a hydrate thereof, and optionally a pharmaceutically acceptable excipient.
9. The compound according to any one of claims 1 to 5, a pharmaceutically acceptable salt thereof or a hydrate thereof, or the pharmaceutical composition according to claim 8 for inhibiting PI3K- in the preparation of an immunosuppressive agent, an mTOR inhibitor, an mTORC1, and an inhibitor Akt-mTOR signaling pathway drugs, drugs that inhibit T lymphocyte proliferation, antitumor drugs, drugs that promote tumor cell apoptosis, drugs that arrest the cell cycle in G1, drugs that prevent organ rejection, drugs that reduce arterial embolism, Use in anti-aging drugs, anti-Alzheimer's disease drugs, anti-inflammatory drugs or antibacterial drugs; in particular, the anti-tumor drugs are for the treatment and/or prevention and/or adjuvant treatment of renal cancer, lymphoma, lung cancer, liver cancer , breast cancer, neuroendocrine cancer, uterine sarcoma or gastric cancer drugs.
10. A coronary stent comprising a pharmaceutical composition comprising the compound according to any one of claims 1 to 5, a pharmaceutically acceptable salt thereof or a hydrate thereof, or the pharmaceutical composition according to claim 8.
11. Inhibition of immunity, inhibition of mTOR, inhibition of mTORC1, inhibition of PI3K-Akt-mTOR signaling pathway, inhibition of T lymphocyte proliferation, anti-tumor, promotion of tumor cell apoptosis, arrest of cell cycle in G1, reduction of arterial embolism, in vivo or in vitro, A method of anti-aging, anti-Alzheimer's disease, prevention of organ rejection, anti-inflammatory or anti-bacterial, comprising the use of an effective amount of the compound according to any one of claims 1 to 5, a pharmaceutically acceptable salt thereof or a hydrate thereof Or the step of the pharmaceutical composition of claim 8.
12. The compound according to any one of claims 1 to 5, a pharmaceutically acceptable salt thereof or a hydrate thereof, or the pharmaceutical composition according to claim 8, which is used as a medicament.
13. The compound according to any one of claims 1 to 5, a pharmaceutically acceptable salt thereof or a hydrate thereof, or the pharmaceutical composition according to claim 8, which is useful for inhibiting immunity, inhibiting mTOR, inhibiting mTORC1, and inhibiting PI3K- Akt-mTOR signaling pathway, inhibit T lymphocyte proliferation, anti-tumor, promote tumor cell apoptosis, arrest cell cycle in G1, reduce arterial embolism, anti-aging, anti-Alzheimer's disease, prevent organ rejection, anti-inflammatory or Antibacterial; in particular, the anti-tumor is for the treatment and/or prevention and/or adjuvant treatment of renal cancer, lymphoma, lung cancer, liver cancer, breast cancer, neuroendocrine cancer, uterine sarcoma or gastric cancer.
14. A method of treating and/or preventing and/or adjuvant treatment of renal cancer, lymphoma, lung cancer, liver cancer, breast cancer, neuroendocrine cancer, uterine sarcoma or gastric cancer, comprising using an effective amount of any one of claims 1 to 5. A step of the compound, a pharmaceutically acceptable salt thereof or a hydrate thereof or the pharmaceutical composition according to claim 8.

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